Unmarked deletion mutants of mycobacteria and methods of using same

ABSTRACT

Disclosed is a recombinant slow-growing mycobacterium comprising at least one mycobacterial gene containing an unmarked mutation, where an “unmarked mutation” is a mutated nucleotide sequence introduced into a mycobacterium where the introduced mutated nucleotide sequence does not contain a selectable marker, such as a gene conferring antibiotic resistance to the recombinant mycobacterium incorporating the mutated nucleotide sequence. Also disclosed is a method for preparing a recombinant slow-growing mycobacterium comprising at least one mycobacterial gene containing an unmarked mutation, as well as a vaccine comprising a recombinant slow-growing mycobacterium having at least one mycobacterial gene containing an unmarked mutation dispersed in a physiologically acceptable carrier. Further disclosed is a method of treating or preventing tuberculosis in a subject comprising administering the vaccine of the present invention in an amount effective to treat or prevent tuberculosis in the subject.

STATEMENT OF GOVERNMENT INTEREST

This invention is supported by NIH Grant Nos. AI26170 and AI33696. Assuch, the U.S. Government has certain rights in this invention.

BACKGROUND OF THE INVENTION

Mycobacterium tuberculosis, the agent of tuberculosis, is the leadingcause of death in adults worldwide (14). The emergence of drug resistantstrains (48) and the problems associated with tuberculosis inHIV-infected populations (18) have brought tuberculosis research to theforefront. The development of genetic techniques to study the biology ofthe organism is an important goal of mycobacterial research.

Considerable effort has gone into the development of allelic exchangemethods to selectively disrupt genes of various mycobacterial species.Several groups have used either small linear DNA fragments (4, 25, 43),long linear DNA fragments (5), or suicidal plasmids, (37, 44) (9, 27,39, 41, 42) to achieve allelic exchange in both fast and slow-growingmycobacteria. Slow-growing mycobacteria such as M. tuberculosis and M.bovis BCG can integrate exogenous DNA into their chromosome by bothillegitimate and homologous recombination (2, 25). Allelic exchange infast-growing mycobacteria such as M. smegmatis is easier than in theslow-growing species; this has led to the idea that the homologousrecombination machinery of slow-growing mycobacteria is ratherinefficient (32).

Thus far, the only mutants constructed in the slow-growing mycobacterialspecies are those with genes disrupted with an antibiotic marker.However, in many cases an antibiotic marker may not be desirable. It maynot be known whether or not a gene is essential and targeted disruptiondoes not let one ascertain essentiality. The failure to obtain a mutantmight be due to the failure of the methodology and not to theessentiality of the gene. Furthermore, the possibility of polar effectsfrom an inserted antibiotic marker can prevent the disruption of anon-essential gene if that gene is located in an operon upstream of anessential gene. Also, there are a limited number of antibioticresistance genes available for use in mycobacteria and making a markedmutation excludes one antibiotic from further consideration. Inaddition, mutants that are potential vaccine candidates should notcontain antibiotic resistance determinants.

An ideal allelic exchange system is one that can be used for theexchange of unmarked deletion alleles as well as alleles with pointmutations. Constructing knockout mutants by in-frame deletions wouldnegate the concerns with using a targeted disruption method. Suchmutants are antibiotic sensitive, cannot revert, and the mutationsshould not be polar on the expression of downstream genes. By extension,the same technique could be used for allelic exchange of pointmutations, allowing for a finer dissection of gene function. Thisallelic exchange methodology, utilizing a plasmid unable to replicate inthe organism of interest and selectable and counter-selectable markers(15), has been successfully used in M. smegmatis (27, 41). The inventorssought to determine if such an allelic exchange methodology wouldreproducibly work for the slow-growing mycobacteria, such as M. bovisBCG and M. tuberculosis.

The inventors describe herein a new mycobacterial suicide plasmid forallelic exchange of unmarked mutations utilizing sacB sucrose counterselection. This counter selectable marker was previously reported towork in mycobacteria, including M. tuberculosis and M. bovis BCG (40)(42) (9). However, the previously described mycobacterial sacB vectorsystems were used for allelic exchange of genes disrupted with anantibiotic resistance marker. The present invention demonstrates thereproducibility of this system for allelic exchange of unmarkeddeletions in the chromosome of M. smegmatis, M. bovis BCG and M.tuberculosis. The inventors have also constructed lysine auxotrophs ofthese three organisms by allelic exchange of lysA, the gene encodingmeso-diaminopimelate decarboxylase, the last enzyme in the lysinebiosynthetic pathway (52). To the best of the inventors' knowledge, thisis the first report of the construction of unmarked deletion mutationsin the genome of slow-growing mycobacteria.

SUMMARY OF THE INVENTION

The present invention discloses a slow-growing recombinant mutantmycobacterium comprising at least one mycobacterial gene containing anunmarked mutation. The invention further provides a method for preparingthe recombinant mutant mycobacterium of the present invention comprisingintroducing a vector into a slow-growing mycobacterium, where saidvector comprises a selectable marker, a counter selectable marker, andan unmarked mutant mycobacterial gene, culturing the slow-growingmycobacterium and selecting for primary recombinants incorporating theselectable marker. The primary recombinants are then cultured, andsecondary recombinants that have lost the counter selectable marker areselected for, followed by isolation of the secondary recombinantsincorporating the desired unmarked mutant mycobacterial gene.

Also provided is a vaccine comprising the slow-growing recombinantmutant mycobacterium of the present invention contained in aphysiologically acceptable carrier, as well as a method of treating orpreventing tuberculosis in a subject comprising administering thevaccine of the present invention in an amount effective to treat orprevent tuberculosis in the subject.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts a map of the suicide vector pYUB657. The vector pYUB657cannot replicate in mycobacteria, but has the ColE1 origin ofreplication for E. coli. The P_(groEL)-sacB cassette is indicated alongwith the sacR regulatory region (50). The vector has the bla gene,conferring resistance to ampicillin in E. coli and the hyg gene,conferring resistance to hygromycin in mycobacteria. This vector is alsoa double cos, PacI-excisable cosmid cloning vector (5). Useful cloningsites are indicated.

FIG. 2 illustrates Southern blots of genomic DNA from four mycobacteriallysA deletion mutants. Panel A depicts genomic DNA from wild-type M.smegmatis mc²155 (Lane 1) and the M. smegmatis auxotroph mc²1493 (Lane2), digested with EcoRi and probed with a 3.3-kb EcoRI fragment fromplasmid pYUB617, encompassing the ΔlysA4 allele. The wild-type fragmentis the expected 4.4-kb, while the mutant has the expected 3.2-kbfragment. Panel B depicts genomic DNA from wild-type BCG substrainPasteur (Lane 1), BCG substrain Pasteur auxotroph mc²1604 (Lane 2),wild-type BCG substrain Connaught (Lane 3), BCG substrain Connaughtauxotroph mc²2519 (Lane 4), wild-type M. tuberculosis H37Rv (Lane 5),and M. tuberculosis H37Rv auxotroph mc²3026 (Lane 6), digested withBssHII and probed with a lysA PCR product obtained from BCG Pasteurwild-type genomic DNA. Digestion of wild-type genomic DNA with BssHIIsplits the lysA gene over two fragments, one which is 1.1-kb in size,the other which is 1.2-kb. Digestion of genomic DNA from the deletionmutants yields the same 1.2-kb fragment seen in wild-type with a 0.9-kbfragment, corresponding to the deletion site, replacing the 1.1-kbfragment. The blots in Panel B show the expected shift in size of the1.1-kb fragment down to 0.9-kb in all three mutants (Lanes 2, 4, and 8).The invariant 1.2-kb fragment shows a lower intensity in the blot due toa lower percentage of homology to the probe, relative to the 1.1 and0.9-kb fragments.

FIG. 3 illustrates the effect of AEC on the growth of wild-type M. bovisBCG, and M. tuberculosis H37Rv. Growth curve data were obtained asdescribed in the Materials and Methods. Panel A illustrates growth of M.bovis BCG substrain Pasteur; Panel B illustrates growth of M.tuberculosis H37Rv. (Key: Basal (7H9 medium), AEC (Basal with 3 mM AEC),Thr (Basal with 3 mM threonine), AEC/Thr (Basal with AEC and threonineat 3 mM each.)

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for yielding recombinantunmarked mutants of mycobacteria, wherein the recombinant mutantmycobacteria comprises at least one mycobacterial gene containing anunmarked mutation. As used herein, an “unmarked mutation” is a mutatednucleotide sequence (i.e., a mutant DNA substrate) that is homologous toand replaces a wildtype nucleic acid sequence of the mycobacteria viahomologous recombination, where said mutant DNA substrate does notcontain a selectable marker, such as a gene conferring antibioticresistance to the recombinant mycobacterium incorporating the mutatednucleotide sequence. The term “recombinant unmarked mutant mycobacteria”as used herein means that the recombinant unmarked mutant mycobacteriumcomprises at least one unmarked mutation, such that the expression orfunction of the mutant DNA substrate incorporated into the recombinantmycobacterium is varied with respect to the non-mutated nucleotidesequence in the parent strain. The method of the present invention isparticularly suited for generating mutants via allelic exchange inMycobacterium tuberculosis complex organisms, preferably strains of M.tuberculosis, M. bovis and Bacille-Calmette-Geurin (BCG), which areslow-growing mycobacteria, as well as in other slow growingmycobacteria, although the method may be used with nontuberculosisfast-growing mycobacteria commonly encountered in biological samplesisolated from human subjects, e.g., M. avium-intracellulare, M.kansasii, M. xenopi, M. scrofulaceum, M. simiae, M. szulgai, M.gordonae, M. gastri, M. smegmatis, and M. chelonae.

The method for preparing a recombinant unmarked mutant of the presentinvention comprises introducing a vector into a slow-growingmycobacterium, where said vector comprises a selectable marker, acounter selectable marker, and a mutant DNA substrate for allelicexchange, then growing the mycobacterium and selecting for primaryrecombinants incorporating the selectable marker, then culturing theprimary recombinants incorporating the selectable marker and selectingfor secondary recombinants that have lost the counter selectable marker,and isolating the secondary recombinants comprising the desired unmarkedmutation. The method of the invention may also be used to producerecombinant unmarked mutant mycobacteria that are fast-growingmycobacteria, including recombinant mutant strains of M. smegmatis or M.avium, but is preferably used to produce recombinant unmarked mutantstrains of slow-growing mycobacteria, and more preferably, recombinantunmarked mutants of M. tuberculosis or M. bovis BCG strains.

The vector of the present invention is a plasmid which is unable toreplicate in mycobacteria (i.e., a suicide plasmid), having a selectablemarker and counter selectable marker on the plasmid backbone. Selectablemarker genes which may be included on the plasmid are well known in theart and include, but are not limited to, genes encoding resistance toantibiotics, including carbenicillin, viomycin, thiostrepton,ampicillin, tetracyline, hygromycin, kanamycin or bleomycin. In apreferred embodiment of the invention, the selectable marker genesincluded on the vector are genes encoding for ampicillin and hygromycinresistance. The counter selectable marker which is included on thevector confers susceptibility to a specific agent, and preferably is oneof the rpsL, pyrF, or sacB genes, and more preferably is the sacB geneencoding for levansucrase and conferring susceptibility to sucrose.

The mutant DNA substrate for allelic exchange may be of any origin, butis preferably from a mycobacterium. In a preferred embodiment of theinvention, the mutated DNA substrate for allelic exchange is from amycobacterium and is homologous to a wildtype nucleic acid sequence ofthe mycobacterium in which it is desired to introduce the mutated DNAsubstrate in lieu of the wildtype nucleic acid sequence.

The DNA substrate for allelic exchange contains the mutation ofinterest, which through allelic exchange, is introduced into andreplaces the homologous region of the mycobacterium nucleic acid. Asused herein, “mutated DNA substrate” refers to the nucleotide sequencefor at least one allele that has been modified by addition, substitutionor deletion of at least one nucleotide, and lacks any selectable marker.In a preferred embodiment of the invention, the mutated DNA substratecomprises a deletion or point mutation of the wildtype nucleic acidsequence. Mutations, including but not limited to deletion, point,substitution, or insertion mutations, may be generated by any number ofmethods known in the art, including but not limited to treatment withrestriction endonucleases, inverse PCR, subcloning techniques and othermethods of in vitro mutagenesis. The wildtype nucleic acid sequence mayencode a protein or polypeptide, and in a preferred embodiment of theinvention encodes an enzyme essential in the biosynthetic pathway of anutrient, structural or cell wall component of the mycobacterium, or anamino acid, such as lysine, leucine, methionine, etc. It is also withinthe confines of the present invention that the wildtype nucleic acid ofthe mycobacterium may comprise an operon or cluster of alleles encodinga number of proteins or polypeptides, or one or more promoters,enhancers or regulators that are involved in the expression andtranslation of mycobacterial proteins and polypeptides. In a preferredembodiment of the invention, the wildtype nucleic acid comprises thelysA gene.

The suicide vector, comprising a selectable marker, a counter selectablemarker, and the mutant DNA substrate for allelic exchange, is introducedto the mycobacteria using any suitable method known in the art,including by electroporation. Primary recombinants incorporating theselectable marker are directly selected for using the appropriate agent,for instance, by exposing the mycobacterium to hygromycin and obtainingHyg^(r) clones where the selectable marker confers resistance tohygromycin. Secondary recombinants that have lost the counter selectablemarker are directly selected for by using the appropriate agent, forinstance, by exposing the mycobacterium to sucrose and obtaining suc^(r)clones where the counter selectable marker is sacB. Once suspectedsecondary homologous recombinants comprising the desired unmarkedmutation are isolated, the unmarked mutation genotype may be confirmedby methods known in the art, such as PCR screening or Southern blotanalysis.

The method of the present invention may be used to generate numerousstrains of auxotrophic recombinant unmarked mutant mycobacteria that areauxotrophic for a particular nutrient or nutrients by reason of thesubstitution via allelic exchange of a wildtype nucleic acid sequence ofa mycobacterium with a mutated DNA substrate. As used herein, the term“auxotrophic recombinant unmarked mutant mycobacterium” is defined as amycobacterium having an unmarked mutation resulting in the nutritionalrequirements of the mycobacterium being altered. For example, someauxotrophic mutants are unable to synthesize amino acids, or may requirespecific amino acids that are not needed by the parental or prototrophicstrain. Specific auxotrophic recombinant unmarked mutant mycobacteria ofthe present invention include slow-growing mycobacteria which areauxotrophic for lysine, although other auxotrophic recombinant unmarkedmutants of slow growing mycobacteria are provided for, includingrecombinant unmarked mutants that are auxotrophic for leucine,threonine, methionine, etc. Preferably, the auxotrophic recombinantmutant mycobacteria are strains of M. bovis BCG or M. tuberculosis, butthe invention is not limited to these species of mycobacteria. In aspecific embodiment of the invention, the auxotrophic recombinantunmarked mutant mycobacteria that is auxotrophic for lysine comprises anunmarked mutation of the lysA gene.

The present invention provides a vaccine comprising an auxotrophicrecombinant unmarked mutant mycobacterium. The invention also provides amethod of treating or preventing tuberculosis in a subject comprisingadministering the vaccine of the present invention in an amounteffective to treat or prevent tuberculosis in the subject. In thisregard, the vaccine containing the recombinant unmarked mutantslow-growing mycobacteria of the present invention may be administeredin conjunction with a suitable physiologically acceptable carrier.Mineral oil, alum, synthetic polymers, etc., are representative examplesof suitable carriers. Vehicles for vaccines and therapeutic agents arewell within the skill of one skilled in the art. The selection of asuitable vaccine is also dependent upon the manner in which the vaccineor therapeutic agent is to be administered. The vaccine or therapeuticagent may be in the form of an injectable dose and may be administeredintramuscularly, intravenously, orally, intradermally, or bysubcutaneous administration.

Further, mycobacteria have well known adjuvant properties and so areable to stimulate a subject's immune response to respond to theirantigens with great effectiveness. Their adjuvant properties areespecially useful in providing immunity against pathogens in cases wherecell mediated immunity is critical for resistance. In addition, themycobacterium stimulates long-term memory or immunity and thus a singleinoculum may be used to produce long term sensitization to proteinantigens. The vaccine of the present invention may be used to primelong-lasting T-cell memory, which stimulates secondary antibodyresponses which will neutralize infectious agents or toxins, e.g.,tetanus and diptheria toxins, pertussis, malaria, influenza, herpesvirus and snake venom.

In addition, the recombinant unmarked mutant mycobacterium of thepresent invention that is auxotrophic for lysine may be used in theconstruction of DAP auxotrophs (peptidoglycan mutants).

The present invention is described in the following Experimental DetailsSection which is set forth to aid in the understanding of the invention,and should not be construed to limit in any way the scope of theinvention as defined in the claims which follow thereafter.

Experimental Details Section

A) Materials and Methods

Bacterial strains and culture methods: The bacterial strains used arelisted in Table 1. The genetic nomenclature for strains bearing anintegrated suicide plasmid (DUP) is as previously described (37). E.coli cultures were grown in LB (Luria-Bertani) broth or on LB agar(DIFCO). Mycobacterial cultures were grown in Middlebrook 7H9 broth(DIFCO) with 0.05% Tween-80® (polyoxyethylenesorbitan monooleate), or on7H9 medium solidified with 1.5% agar or on Middlebrook 7H10 or 7H11media (DIFCO). All cultures were incubated at 37° C. All Middlebrookmedia were supplemented with 0.2% (v/v) glycerol and with 1X ADS (0.5%bovine serum albumin, fraction V (Boehringer Mannheim), 0.2% dextrose,and 0.85% NaCl) for M. bovis BCG and M. tuberculosis cultures. Basalmedia were 7H9 and 7H10 supplemented as described above. Sucrose wasused in the medium at a concentration of 2% (w/v), added after themedium was autoclaved and cooled to 55° C. Casamino acids(acid-hydrolyzed casein, DIFCO) was used at a concentration of 0.2 %(w/v). Individual amino acids were obtained from Sigma Chemical (St.Louis, Mo.) and used at a concentration of 40 μg/ml, unless indicatedotherwise. The lysine analog S-(β-aminoethyl)-L-cysteine (AEC) wasobtained from Sigma Chemical, dissolved in water and used at aconcentration of 3 mM. When required, the following antibiotics wereused at the specified concentrations; carbenicillin (50 μg/ml; E. coli),kanamycin A monosulfate (25 μg/mil; E. coli, M. smegmatis, M. bovisBCG), hygromycin B (50 μg/ml; E. coli, M. bovis BCG, and M.tuberculosis, 150 μg/ml; M. smegmatis). Hygromycin B was purchased fromBoehringer Mannheim (50 mg/ml in phosphate buffered saline), all otherantibiotics were purchased from Sigma Chemical. It was often found thatpYUB412 and pYUB405-based plasmids were only stable in E. coli usingboth carbenicillin and hygromycin at 50 μg/ml in solid and liquid media.M. smegmatis plates were incubated for 3-5 days, while M. bovis BCG andM. tuberculosis plates were incubated for 3-4 weeks. M. smegmatis liquidstarter cultures were inoculated from plates into 10 ml of medium in 30ml plastic media bottles, grown for 1-2 days on a shaker platform at 100rpm and then subcultured 1:100 in fresh media within 250 ml glass baffleflasks. M. bovis BCG and M. tuberculosis starter cultures wereinoculated using 1 ml frozen stocks in 10 ml of media in 30 ml plasticmedia bottles and incubated for 5-7 days on a shaker platform at 100rpm. Larger cultures were inoculated from the starter cultures at a 1:50dilution in 50 ml or 100 ml of medium within 490 cm² roller bottles(Coming) and incubated on a roller apparatus at 8 rpm for 5-7 days. Forgrowth curves, mid to late exponential phase cultures were centrifuged,washed with fresh media lacking supplements, and the cells resuspendedappropriately and inoculated into test media. Samples of M. tuberculosisand BCG cultures were mixed 1:1 with 10% phosphate-buffered formalin andfixed for at least 1 hour prior to spectrophotometric measurement atO.D.₆₀₀.

DNA methodologies: DNA manipulations were done essentially as previouslydescribed (29). The plasmids used in this study are listed in Table 2.Plasmids were constructed in E. coli HB101 or DH5α cells and prepared byan alkaline lysis protocol (22). Plasmids used for recombination werepurified using Qiagen columns as recommended by the manufacturer(Qiagen, Inc., Chatsworth, Calif.). DNA fragments used for plasmidconstruction were purified by agarose gel electrophoresis and recoveredby absorption to glass fines (GeneClean, Bio 101, Vista, Calif.).

Genomic DNA was prepared either as previously described (23) or by amodified guandinium thiocyanate protocol (34). Briefly, the cells from a10 ml culture are lysed with 1.3 ml of a 3:1 mixture of chloroform:methanol. The lysate is mixed with 1.3 ml of Tris-equilibrated phenoland a 2 ml of GTC solution (4 M guandinium thiocyanate, 0.1 M Tris pH7.5, 0.5% sarcosyl, with β-mercaptoethanol added to a finalconcentration of 1% prior to use). The upper phase is collected aftercentrifugation and the genomic DNA precipitated with isopropanol.Southern blotting and hybridization were done as previously described(37). Oligonucleotides for sequencing and PCR were synthesized by theAlbert Einstein College of Medicine oligonucleotide synthesis facility.

Cloning and sequencing of the M. smegmatis lysA operon: The inventorsused a library of genomic DNA from wild-type M. smegmatis mc²155constructed in the cosmid vector pYUB412 to clone the lysA gene. Thevector pYUB412 is an integration-proficient, PacI-excisable cosmidvector (6). This cosmid vector has the mycobacteriophage L5 attachmentsite (attP), the L5 integrase gene (int), and the hyg gene, conferringresistance to hygromycin. This vector efficiently integrates into themycobacteriophage L5 attachment site (attB) of the mycobacterialchromosome and is stable (28). The pYUB412::mc²155 library waselectroporated into the strain MCK3037, a lysine auxotrophic mutant ofmc²155 generated by EMS mutagenesis (33). Transformants were selected on7H10 media lacking lysine and Lys⁺ clones screened for the hygromycinresistance marker carried on the cosmid vector backbone. One Lys⁺Hyg^(r) clone was chosen for study and the genomic DNA insert within theintegrated cosmid recovered by λ in vitro packaging (GigaPak III,Strategene). The recovery procedure is as follows: the library insertDNA is flanked by PacI restriction endonuclease sites present in thecosmid vector, and since PacI sites do not exist in mycobacterialgenomic DNA (26), PacI digestion of the genomic DNA will release thecosmid insert DNA. This DNA fragment is re-packaged into PacI-digestedarms of the cosmid vector pYUB412 by λ in vitro packaging, and a newcosmid (pYUB601) with the insert recovered in E. coli. The cosmidpYUB601 insert DNA was subcloned to a 4.4-kb EcoRI fragment bearing thelysA gene in plasmid pYUB604. The plasmid pYUB604, and two subclones,pYUB605 and pYUB607, were templates for DNA sequencing using the AppliedBiosystems Prism Dye Terminator Cycle Sequencing Core kit with AmpliTaqDNA polymerase (Perkin Elmer) and an Applied Biosystems 377 automatedDNA sequencer. Sequence data for both strands of the lysA operon of M.smegmatis were obtained from these subclones and by primer walking.

Construction of sacB suicide vector pYUB657: A 2.5-kb PstI fragment fromthe E. coli sacB vector pVCD442 bearing sacB and its upstream regulatoryregion sacR, were subcloned into the PstI site of the shuttle vectorpMV261 downstream of the mycobacterial groEL (Hsp60) promoter, yieldingthe plasmid pYUB631. A 3.5-kb NotI-NheI fragment from pYUB631, bearingP^(groEL)-sacB was cloned into the cosmid vector pYUB405, resulting inthe final construct, pYUB657 (see FIG. 1). The vector pYUB405 is aPacI-excisable cosmid vector unable to replicate in mycobacteria andencodes resistance to ampicillin and hygromycin (6).

Construction of the M. smegmatis ΔlysA4 suicide plasmid pYUB618: Theplasmid pYUB604 was used as the template in an inverse PCR reaction toproduce a deletion within the lysA gene. Oligonucleotide primers Pv44(5′-CCCGTCGTACGTACGAACCAGGTTGCGC-3′) (SEQ ID NO:1) and Pv45(5′-CGAGTCGATACGTACTGCTGTGCCGCCC-3′) (SEQ ID NO:2) were used at 50 pmoleach in an inverse XL-PCR reaction in a Perkin Elmer 9600 temperaturecycler with the following program: 95° C./5 min, 1 cycle; 93° C./1min-68° C./5 min, 16 cycles; 93° C./1 min-68° C./5 min with ΔTh=15 sec,12 cycles; 72° C./30 min. The reaction produced a 7.7-kb fragment with a1.2-kb deletion within the lysA ORF (spanning nt positions 2051 . . .3251 of GenBank accession AF126720) marked with a unique SnaBI site. ThePCR product was gel purified, digested with SnaBI and self-ligated toyield the plasmid pYUB617. A 3.2-kb EcoRI fragment from pYUB617 bearingthe ΔlysA4 allele was cloned into the PacI sites of the mycobacterialsacB suicide vector pYUB657, resulting in the M. smegmatis ΔlysA4suicide plasmid pYUB618.

Construction of the M. bovis BCG/M. tuberculosis ΔlysA5:: res suicideplasmid pYUB668: The lysA gene of M. tuberculosis was originally clonedand sequenced by Anderson et al. (3). The plasmid pET3d.lysA containsthe lysA gene of M. tuberculosis strain Erdman cloned by PCR usingprimers designed from the previously published sequence (3) (16). A1.3-kb XbaI-BamHI fragment bearing the lysA gene was cloned frompET3d.lysA into the same sites in PKSI⁺ to produce pYUB635. This plasmidwas used as the template in an inverse PCR reaction with theoligonucleotide primers Pv7: (5-′GATAGCGGTCACGCGTCTCGTGCGCGGTGGA-3′)(SEQ ID NO:3) and Pv8 (5-TCCGTACGATACGCGTCAGCCACATCGGTTCG-3′) (SEQ IDNO:4) to generate a 95-bp deletion within the lysA gene marked with aunique MluI restriction endonuclease site. The inverse XL-PCR reactionwas done using a Perkin Elmer 9600 temperature cycler and the programdescribed above for plasmid pYUB617. The resulting 4.1-kb PCR productwas gel-purified, digested with MluI and self-ligated to yield theplasmid pYIJB636. The lysA deletion was marked with the aph gene,conferring kanamycin resistance, by insertion of a specialized aphcassette via the unique MluI site to yield pYUB638. This specializedcassette has an aph gene flanked by two γδ resolvase sites from the E.coli transposon γδ (Tn1000) (20). The presence of the resolvase sitesmakes it possible to excise the antibiotic marker by expressing the γδresolvase in mycobacteria after the cassette has been inserted into themycobacterial chromosome (8). In the present case, however, theres-aph-res marker was removed from pYUB638 by resolvase excision in E.coli DH5α prior to introduction into mycobacteria (see below).

To include more DNA on both sides of the M. tuberculosis ΔlysAconstruct, cosmid cosY373 from the Sanger Centre M. tuberculosis H37Rvgenome sequencing project (12) was used. An 11-kb SnaBI fragment fromcosY373, containing lysA situated in the middle, was subcloned into theEcoRV site of pKSI⁺ to yield plasmid pYUB659. To replace the wild-typelysA allele in pYUB659 with the ΔlysA::res-aph-res allele constructedabove in pYUB638, the inventors exchanged an internal NheI-BglIIfragment of lysA encompassing the deletion region between these twoplasmids. Because there is an additional NheI site at the 5′ end of theres-aph-res cassette, this exchange resulted in an additional deletionof 236-bp within the lysA gene. The resulting plasmid, pYUB665, containsa deletion within lysA totaling 331-bp and the res-aph-res cassette.Plasmid pYUB665 was passaged in E. coli DH5α (which has a γδ elementcapable of excising the aph gene from the ΔlysA::res-aph-res allele) andisolated a Kn^(s) derivative, plasmid pYUB667. DNA sequence analysis ofpYUB667 showed that the aph cassette was absent and a single res siteremained that was in-frame with respect to the lysA open reading frame.The mutant lysA allele in pYUB667 is designated ΔlysA5::res and has atotal deletion of 331-bp of an internal portion of the lysA gene, butwith the addition of the 136-bp res site, the net change in size ofΔlysA5::res compared to wild-type is a decrease of 195-bp. To producethe final suicidal plasmid for allelic exchange in M. bovis BCG and M.tuberculosis, a 8.4-kb HpaI fragment from pYUB667 was cloned into thePacI sites of the sacB suicidal vector pYUB657, resulting in plasmidpYUB668. This plasmid has approximately 4-kb of DNA flanking each sideof the ΔlysA5::res allele.

Electroporation of mycobacteria: M. smegmatis was electroporated aspreviously described (37). M. bovis BCG and M. tuberculosis wereelectroporated as per M. smegmatis, except that all manipulations weredone at room temperature instead of on ice and the expression stepproceeded overnight for approximately 12 hours prior to plating.

Nucleotide sequence accession number: The DNA sequence of the 4462 bpEcoRI fragment encoding the M. smegmatis lysA gene was submitted toGenBank and assigned the accession number AF126720.

B) Results

Allelic exchange methodology: The basic procedure for making mutantswith the sacB suicidal vector pYUB657 (FIG. 1) is a two-step allelicexchange (15) (38). A suicidal recombination plasmid is electroporatedinto cells and primary recombinants selected upon hygromycin medium.Since the plasmid cannot replicate, any hygromycin resistant clones musthave integrated the plasmid into the chromosome by a single crossoverevent. Because of the presence of the sacB gene on the pYUB657 vectorbackbone, the Hyg^(r) clones are also sensitive to sucrose (Suc^(s)).Plasmid integration at the desired locus results in a tandem duplication(given the designation DUP) of the cloned region with the vector DNA inthe middle. One such DUP clone is grown to saturation in supplementedmedium, during which time individuals within the population undergo asecond homologous recombination event between the duplicated regions. Inthis event, the plasmid vector loops out and is lost along with the hygand sacB genes, leaving behind either the wild-type or mutant allele,depending upon which side of the mutation the second recombination eventoccurred. This second recombination event occurs at a low frequency,thus there must be a selection for the desired secondary recombinants.To select these clones one takes advantage of the loss of the sacB gene;any clone losing the plasmid is now sucrose resistant (Suc^(r)). Theculture is plated on supplemented media containing sucrose to kill anyclones that did not undergo a second recombination event. The sucroseresistant clones are then screened for hygromycin sensitivity and themutant phenotype.

Cloning of the mycobacterial lysA genes: The present system byconstructing lysine auxotrophs via deletion of the lysA gene, encodingmeso-diaminopimelate decarboxylase, in M. smegmatis, M. bovis BCG, andM. tuberculosis. The lysA gene of M. tuberculosis was already availableand could also be used for allelic exchange in M. bovis BCG due to theconservation of DNA sequences between the two species, however, the lysAgene of M. smegmatis was not available. The M. smegmatis lysA gene andresident operon was cloned as described in the Materials and Methods.The lysA gene of M. smegmatis is 1424-bp in length and shares 77%homology with the lysA gene of M. tuberculosis, while the two LysAproteins share a 80% identity (17). The structure of the lysA operon isconserved between several mycobacteria and the related organismCorynebacterium glutamicum. In M. tuberculosis, the gene order is: argS(arginyl-tRNA synthetase), lysA(meso-DAP decarboxylase), hdh (homoserinedehydrogenase), thrC (threonine synthase), PGRS-17 (poly GC-rich repeat17), and thrB (threonine kinase) (http). The sequence from M. smegmatisspans upstream of args through the hdh gene. A similar argS-lysA operonarrangement is seen for M. leprae (37) and Brevibacterium glutamicum(renamed Corynebacterium glutamicum) (35). The hdh gene product supplieshomoserine, the precursor for Met and Thr biosynthesis (30); while thethrC and thrB genes are responsible for threonine synthesis (36).

Construction of an unmarked lysA deletion mutant of M. smegmatis: M.smegmatis mc²155 was electroporated with the ΔlysA4 suicidal plasmidpYUB618 (see Materials and Methods for plasmid construction) andobtained an average of 15 Hyg^(r) clones per transformation, withprimary recombination efficiencies of 10⁻⁵ (see Table 3). Two culturesof one strain, mc²1492, were grown to saturation in 7H9/lysine media anddilutions plated onto 7H10/lysine medium supplemented with sucrose.Sucrose resistant clones were obtained at a frequency of 10⁻⁴, and 100clones from each set were screened for Suc^(r), Hyg^(s), and auxotrophy.Three basic phenotypes were found: Suc^(r)/Hyg^(r)/prototrophic,Suc^(r)/Hyg^(s)/prototrophic, and Suc^(r)/Hyg^(s)/auxotrophic (see Table4, exps. 1 and 2). The largest group was theSuc^(r)/Hyg^(r)/prototrophic class and likely resulted from inactivationof the sacB gene, since the clones were still resistant to hygromycinand did not appear to have arisen from a secondary recombination event.The other two Suc^(r) classes were Hyg^(s) and appeared to result fromsecondary recombination events; the first class retained the wild-typeallele, while the second class retained the mutant allele and wereauxotrophic for lysine. One mutant was given the designation mc²1493 andallelic exchange of lysA confirmed by Southern blot (see FIG. 2, panelA). The mutant grows equally well on defined 7H9 medium supplementedwith lysine or on complex media (7H9 supplemented with casamino acids orLB medium).

Construction of an unmarked lysA deletion mutant of M. bovis BCGsubstrain Pasteur: The suicide plasmid pYUB668 (see Materials andMethods) was used to construct an unmarked, in-frame deletion of lysA(ΔlysA5::res) in the genome of M. bovis BCG substrain Pasteur. Afterelectroporation of BCG substrain Pasteur with the suicide plasmid, anaverage of 5 Hyg^(r) clones were obtained per transformation with aprimary recombination efficiency of 10⁻⁴ (see Table 3). Several Hyg^(r),Suc^(s) clones were screened by PCR to determine which of the primaryclones were homologous recombinants. Three out of four clones examinedhad incorporated the suicide plasmid pYUB668 at the lysA locus, whilethe fourth appeared to be the result of an illegitimate recombinationevent (data not shown). Two clones were chosen for further study,mc²1601 (DUP3) and mc²1602 (DUP4) both of which had integrated pYUB668at lysA but had differed in the orientation of the duplication (seeTable 1). The two strains were grown to saturation in 7H9 mediasupplemented with lysine, methionine, and threonine and then plated uponthe same type of media containing sucrose. This combination of aminoacids was used to ensure that any unforeseen polar effect of theΔlysA5::res allele on the downstream Met and Thr biosynthetic geneswould not prevent the isolation of mutants. The results of the sucroseselection are shown in Table 4, exp 3 and 4. Suc^(r) clones wereobtained at a frequency of 10⁻⁴ and observed the same three classes ofsecondary recombinants that we saw in the M. smegmatis experiments.Allelic exchange was confirmed in strain mc²1604, a mutant derived fromDUP3 strain mc²1601 (see Southern blot, FIG. 2, panel B). The auxotrophmc²1604 does not revert, and no suppression was observed in twoindependent cultures of 5×10⁹ CFU each.

The kinetics of allelic exchange of lysA in M. bovis BCG substrainPasteur was surprisingly similar to that of M. smegmatis promptingexamination of the reproducibility of this system. Sucrose selection wasrepeated with M. bovis BCG substrain Pasteur DUP3 strain mc²1601 usingcultures grown in Basal medium or media supplemented with Lys,Met+Thr+Lys, or casamino acids (acid-hydrolyzed casein). Suc^(r) cloneswere obtained from each of the respective cultures at a frequencysimilar to those seen in the previous experiment with mc²1601 (See Table4, exps. 5 through 8, compare to exp. 3). The distribution of the threephenotypic classes in the Suc^(r) population was also similar exceptthat no lysine auxotrophs were obtained from cultures grown in Basalmedium lacking lysine (as expected) or, surprisingly, casamino acidsmedium (Table 4, exps. 5 and 8).

Using allelic exchange to distinguish homologous from illegitimateprimary recombinants: When using the two-step allelic exchangemethodology with the slow-growing mycobacteria, it is important toidentify primary recombinants that resulted from illegitimaterecombination and those which resulted from homologous recombination.This can be done by PCR screening (as we did for the above experiment)or Southern blot, although these screening methods are difficult whenusing large recombination substrates. The inventors reasoned it shouldbe possible to distinguish between the two types of recombinants byobserving the phenotypic frequencies in the pool of Suc^(r) secondaryclones. Presumably, any primary recombinant resulting from a homologousintegration of the plasmid at lysA would be able to undergo a secondrecombination event and loop out the plasmid, while a recombinant thathad integrated the plasmid via illegitimate recombination would beunable to do the same. Any Suc^(r) clones arising from an illegitimaterecombinant would result from inactivation of the sacB gene as seenabove and all these clones should also be Hyg^(r).

This idea was tested in a series of lysA allelic exchange experimentswith BCG substrain Connaught. Electroporation of BCG Connaught with thesuicide plasmid pYUB668, yielded an average of 2 Hyg^(r) clones perelectroporation for a primary recombination efficiency of 10⁻³ (seeTable 4). 7 Hyg, Suc^(s) BCG Connaught::pYUB668 primary recombinantswere chosen, grown in media supplemented with lysine and plated forsucrose resistant clones. The Suc^(r) clones were screened forhygromycin sensitivity and auxotrophy (see Table 4, exps 9 through 15).Three of the seven primary recombinants (clones 3, 9, and 10) gave riseto similar phenotypic populations as that seen for M. bovis BCGsubstrain Pasteur DUP strains mc²1601 and mc²1602 (compare results inTable 4). Therefore, these three primary clones (3, 9, and 10) werehomologous primary recombinants. Two clones (2 and 11) yielded onlySuc^(r), Hyg^(r), prototrophs, while the remaining clones (4 and 8)yielded a majority of Suc^(r), Hyg^(r), prototrophs and a small numberof Suc^(r),Hyg^(s), prototrophs (Table 4). These four primary clones (2,4, 8, and 11) were classified as illegitimate recombinants. One BCGsubstrain Connaught lysine auxotroph, derived from clone 3 wasdesignated strain mc²2519, and allelic exchange confirmed by Southernblot (see FIG. 2, panel B).

Construction of an unmarked, in-frame lysA deletion mutant of M.tuberculosis strain H37Rv: The same methodology and suicide plasmid,pYUB668, described above was used to construct a lysA deletion mutant ofM. tuberculosis H37Rv. Primary recombination efficiencies were observedthat were similar to those observed in experiments with BCG substrainPasteur (see Table 3). Six Hyg^(r), Suc^(s) primary recombinants werechosen, grown in lysine supplemented medium and plated for sucroseresistant recombinants. All 6 primary recombinants gave rise tophenotypic populations similar to the results seen with the BCG mc²1601DUP3 strain grown in Basal medium in Table 4, exp. 5 (data not shown).It was concluded that these primary clones were all likely homologousrecombinants, but that something was wrong with the system since we didnot isolate any auxotrophs. The sucrose selection was repeated with twoof these primary recombinant strains, mc²2998 and mc²2999, grown inseveral types of media: Basal, Lys, Met+Thr+Lys, and casamino acid (seeTable 4, exps. 16 through 23). The frequency of sucrose resistance wasin the range of 10⁻⁵ to 10⁴ (see Table 4). Again, auxotrophs were notobtained and it was confirmed that the phenotypic frequencies within theSuc^(r) population were similar to the failure to isolate Lys-BCGmutants on Basal medium (compare Table 4, exps. 17 and 18 with exp. 5).Furthermore, the results from the M. tuberculosis primary recombinantswere unlike the results obtained with the BCG Connaught illegitimateprimary recombinants. Thus, these results suggested that the primaryrecombinants were indeed homologous, but for some reason any auxotrophsresulting from a secondary recombination event were nonviable.Apparently, the media could not support the growth of a M. tuberculosislysine auxotroph. It was decided to determine if the inability toisolate a lysine auxotroph of M. tuberculosis was due to the inabilityof the organism to transport lysine.

Transport of lysine in mycobacteria: To investigate lysine transport inM. tuberculosis, the toxic lysine analog S-(β-aminoethyl)-L-cysteine(AEC) was used. AEC is transported via lysine importers; the lysinepermeases of E. coli (LysP), and Corynebacterium glutamicum (LysI) wereidentified using AEC-resistant mutants (46, 49). AEC inhibitsaspartokinase, the enzyme catalyzing the first step of the aspartateamino acid family pathway responsible for the synthesis of Met, Thr,Ile, Lys, and DAP (meso-diaminopimelate), the latter begin a componentof the cell envelope peptidoglycan and the precursor to lysine (45)(24). AEC alone is capable of inhibiting the growth of E. coli, butrequires the addition of threonine to inhibit the growth of C.glutamicum (45). Presumably, full AEC sensitivity in corynebacteriarequires repression of the threonine branch of the pathway by threonine.

The growth curves of M. tuberculosis strain H37Rv and BCG substrainPasteur in media with or without AEC and Thr are shown in FIG. 3. Amolar concentration of 3 mM was used for AEC and Thr, a concentrationthat is close to the 40 μg/ml used for amino acid supplementation in theinventors' studies. As seen in FIG. 3, panels A and B, neither AEC orThr alone have an inhibitory effect upon the growth of the two species,however the combination of the two does inhibit growth, with BCGexperiencing the greatest inhibition compared to M. tuberculosis. Oneinterpretation of the results of this experiment is that lysine uptakeis not as efficient in M. tuberculosis compared to BCG. The BCG lysineauxotrophic mutant mc²1604 does not grow well in media supplemented withlysine at concentrations below the standard concentration of 40 μg/ml(data not shown). This suggests that a decrease in transport efficiencyof M. tuberculosis compared to that of BCG might preclude isolation of aM. tuberculosis lysine auxotroph. Since the inability to isolate alysine auxotroph of M. tuberculosis might be due to inefficient lysinetransport by the organism, another attempt was made using media withincreased amounts of lysine.

Identification of media that support the growth of a M. tuberculosisH37Rv lysine auxotroph: Allelic exchange with the M. tuberculosisprimary pYUB668 homologous primary recombinant strain mc²2999 wasrepeated using modified media with increased amounts of lysine.Experiments utilizing media containing lysine at 200 μg/ml, or 200 μg/mlwith 0.05% Tween-80, or lysine at 1 mg/ml did not yield any auxotrophs(Table 4, exps. 24-26). However, auxotrophic mutants were isolated whenmedia containing lysine at 1 mg/ml with 0.05% Tween-80 was used (Table4, exp. 27). The mutants produce colonies that are much smaller thanwild-type and were easily identified on the sucrose selection plates(see Table 4, exp 27).

One mutant was designated mc²3026 and allelic exchange of lysA wasconfirmed by Southern blot (see FIG. 2, Panel B). No reversion orsuppression was seen in 3×10⁹ CFU. The mutant grows slowly, requiringapproximately 4-5 weeks to form a large colony on solid media and has anapproximate doubling time of 48 hours in liquid medium (data not shown).Surprisingly, the mutant can grow on 7H10 solid media supplemented withcasamino acids and also grows on 7H11 (supplemented with casitone, apancreatic digest of casein), but requires high concentrations of lysineif lysine is the sole supplementation. It has an absolute dependencyupon Tween-80 regardless of the type of solid media.

C) Discussion

Several groups have demonstrated the use of suicide plasmids for allelicexchange in fast and slow-growing mycobacteria. The most efficient arethose systems using a counter selectable marker; for mycobacteria,workers have successfully used rpsL (37, 44), pyrF (27), and sacB (42).The most promising counter selectable system for the slow-growingmycobacteria is sacB, which confers sensitivity to sucrose.Methodologies using sacB were used for the targeted disruptions of ureCin BCG (42) and M. tuberculosis (39); and the erp gene of BCG and M.tuberculosis (9).

It was decided to construct a new sacB suicidal vector, pYUB657, andtest it for the construction of unmarked, in-frame deletion mutants inthe slow-growing mycobacteria. These studies provided an opportunity toexamine homologous recombination in the mycobacteria from a practicalstandpoint. The bane of allelic exchange in slow-growing mycobacteriahas been the propensity with which these organisms incorporate exogenousDNA into their genome via illegitimate recombination (25) (2, 32).Allelic exchange in M. smegmatis is relatively easy, and this speciesdoes not appear integrate DNA via illegitimate recombination. Severalworkers have suggested that the homologous recombination machinery israther inefficient in the slow-growing mycobacteria. It is generallybelieved that illegitimate recombination occurs at a higher frequencythan homologous recombination in the slow-growing mycobacteria, but thisdoes not necessarily mean that homologous recombination is defective inthese organisms (32).

In any allelic exchange technique with the slow-growing mycobacteria, itis important to distinguish homologous primary recombinants fromillegitimate recombinants; in a method of the present invention, thiswas done by observing the frequencies of the phenotypes in the Suc^(r)populations. The inventors's experiments with BCG substrain Connaughtand M. tuberculosis pYUB668 recombinants showed that one using thepresent method could reproducibly determine if they had a primaryhomologous recombinant, obtain the mutant or discover that the mutationwas not permitted, all at once. The illegitimate pYUB668 recombinants ofBCG substrain Connaught were apparently unable to undergo a secondrecombination event, since virtually all of the Suc^(r) clones were sacBinactivated clones. A small number of Suc^(r) Hyg^(r) clones fromConnaught::pYUB668 clones 4 and 8 may have arisen from deletions withinthe integrated plasmid.

The results of this work suggest that homologous recombination in M.bovis BCG and M. tuberculosis is as efficient as that in M. smegmatis.First, the frequency of integration of suicidal plasmids into thechromosomes of the fast and slow-growers is similar, within the 10⁻⁴ to10⁻⁵ range (except for BCG-Connaught which was 10⁻³; this might be aninflated value however, due to an unusually low electroporationefficiency with the control vector pYUB412). While the number of primaryrecombinants obtained in BCG and M. tuberculosis is often less than thatobtained in M. smegmatis, the differences in the number of primaryrecombinants and recombination frequencies are small, and theelectroporation frequencies are at best, only an approximation. It issuspected that any significant differences in primary recombinationfrequencies between slow-growers and M. smegmatis likely reflect adifference in DNA entry into the cells, since it is generally agreedthat higher electroporation efficiencies are possible with M. smegmatisthan with the slow-growers.

The recombination frequencies for the slow-growing mycobacteria includesboth homologous and illegitimate recombinants, thus a direct comparisonbetween the frequencies of primary recombination in fast andslow-growing mycobacteria may not be valid. However, more illegitimaterecombination may occur with linear DNA than that which occurs withplasmid DNA. Electroporation of digested, linear insert DNA from therecombination plasmids of the present invention into BCG yielded 10 foldmore clones than electroporation with the plasmids, but all clones wereillegitimate recombinants (data not shown). In addition, we rarelyobtained hygromycin resistant clones were rarely obtained when the sacBsuicide vector pYUB657 lacking a DNA insert for recombination waselectroporated into BCG or M. tuberculosis (data not shown).

Comparing homologous recombination frequencies among these three speciesis more straightforward when one examines the frequencies of secondaryrecombination events. When cultures were subjected to sucrose selection,sucrose resistant clones were obtained in the range of 10⁻⁴ to 10⁻⁵ forall three species, the same as the frequency seen for the primaryrecombination of the plasmid into the chromosome. In the sucroseresistant population, three phenotypic classes were observed, two ofwhich resulted from a recombination event and one that the inventorsbelieve did not. The latter class, the Suc^(r), Hyg^(r) prototrophs weredesignated “sacB inactivated” clones, since they were still hygromycinresistant. Inactivation of sacB at a similar frequency to that observedin this study has been noted previously (42). Counter-screenable markerscan be inactivated at an approximate frequency of 10⁻⁵ in M. smegmatisby the action of mobile insertion elements (11). A similar phenomenon,at a lower frequency, has been seen using the rpsL system for allelicexchange in M. smegmatis (37).

In this study, mutants were constructed with a deletion in lysA,conferring a lysine auxotrophic phenotype. Unexpectedly, the lysineauxotrophs described herein have different lysine requirements. The M.smegmatis mutant is the most flexible in its requirements, growing onchemically defined media supplemented with lysine as well as mediumsupplemented with casamino acids. In contrast, auxotrophs of BCG Pasteurcould not be isolated using casamino acids-containing media, even thoughthe compositional analysis of the casamino acids used in this studyshowed that the media should have a lysine concentration that isthree-fold greater than the amount required for the BCG lysineauxotrophs (13). Neither the BCG Pasteur or Connaught lysine auxotrophsare able to grow on solid media if casamino acids or casitone (apancreatic digest of casein) is used as the source of lysine. Previouslystudied Met, Ile-Val, and Leu auxotrophic mutants of BCG can grow on allof these media, unlike the BCG lysine auxotrophs described in this study(31) (25). In more recent work with transposon mutagenesis of BCG; therewere attempts to assay the efficiency of mutagenesis by screening foramino acid auxotrophy (7). The only mutants that were obtained were Leuauxotrophs, as isolated previously. This led to some concern that thetransposition mechanism might not be random which would be detrimentalto a mutagenesis system (6). However, all of these attempts utilizedmedia containing casein preparations. Under such conditions, lysineauxotrophs would not be isolated. It is possible that the caseinphenomenon described here is more widespread and could explain thedearth of auxotrophs in the above experiments. The inventors arecurrently investigating why the BCG lysine auxotrophs fail to grow onmedia containing casein.

Lysine auxotrophs of M. tuberculosis H37Rv were not isolated until mediawith a high concentration of lysine and 0.05% Tween-80 was used. As inthe case for BCG, M. tuberculosis mutants could not be isloated usingcasamino acids, however, once a mutant was obtained, the inventors foundthat it could grow on casamino acids media or casitone, as long as therewas Tween-80 in the media. Since the M. tuberculosis mutant is dependentupon the presence of Tween-80, the inventors assume that the failure toobtain a mutant using casamino acids media was due to the absence ofTween in the selection media. It is important to note that Tween-80 doesnot allow the BCG auxotrophs to form colonies of casamino acids media.Based upon the AEC toxicity data, it can be concluded that M.tuberculosis H37Rv does not transport lysine as effectively as BCG.Alternatively, since AEC toxicity requires transport of threonine aswell, the AEC results could be explained by inefficient threoninetransport. However, the high lysine requirement of the mutant and thedependency upon Tween-80 would support the former conclusion, sinceTween-80 is believed to increase the permeability of the mycobacteriacell envelope (21). The primary phenotypic difference between the BCGand the M. tuberculosis mutants is that the BCG mutants require lysinesupplementation alone, while the M. tuberculosis mutant requiresTween-80 along with either lysine at high concentration or casaminoacids.

The auxotrophic mutants obtained herein will be useful in a variety ofapplications. The BCG and M. tuberculosis lysine mutants may be usablefor the construction of DAP auxotrophs (peptidoglycan mutants), as theinventors have done for M. smegmatis (37). A series of vectors bearingthe lysA gene are also being developed that could be used for theexpression of foreign antigens in the BCG auxotrophs; the presence ofthe lysA gene would maintain the plasmids in vivo in the absence ofantibiotic selection. The behavior of the BCG mutants in animals isbeing tested in the hope that they could be used in HIV infectedpopulations as a safer alternative to live, wild-type BCG vaccine. Onemajor goal of mycobacterial research is the development of attenuatedstrains of M. tuberculosis that could be used as potential vaccinestrains. Such mutant strains would be unable to grow in a host, or growonly for a short time, lasting long enough to prime the immune system.To this end, the inventors are currently examining the growth kineticsof the M. tuberculosis auxotroph in animal models.

TABLE 1 Strains used in this study Strain Description Reference E.coliK-12 HB101 F- Δ(gpt-proA)62 leuB1 glnV44 ara-14 lacY1 hsdS20 (10) rpsL20xyl-5 mtl-1 recA13 DH5α F- [φ80dΔlacZM15]Δ(lacZYA-argF)U169 deoR recA1(19) endA1 hsdR17 glnV44 thi-1 gyrA96 relA1 M. smegmatis mc²155 ept-1(47) mc²1492 ept-1 DUP2 [(argS ΔlysA4 hdh′)*pYUB657*(argS lysA This workhdh)] mc²1493 ept-1 ΔlysA4 This work M. bovis BCG Pasteur Vaccine strainStatens Seruminstitut mc²1601 Pasteur DUP3 [(argS lysA hdhthrC)*pYUB657*(argS This work ΔlysA5::res hdh thrC′)] mc²1602 PasteurDUP4 [(argS ΔlysA5::res hdh This work thrC)*pYUB657*(argS lysA hdhthrC)] mc²1604 Pasteur ΔlysAS::res This work Connaught Vaccine strainAECOM mc²1618 Connaught::pYUB668 homologous primary recombinant, Thiswork clone 3 mc²2519 Connaught ΔlysA5::res This work M. tuberculosisH37Rv Virulent AECOM mc²2998 H37Rv::pYUB668 homologous primaryrecombinant, This work clone 1 mc²2999 H37Rv::pYUB668 homologous primaryrecombinant, This work clone 2 mc²3026 ΔlysA5::res This work

Name Description Reference pKSI⁺ Ap^(r), high copy number cloning vectorStratagene pMV261 Km^(r), E. coli-mycobacterial shuttle vector (51)pET3d.lysA M. tuberculosis Erdman lysA gene cloned into pET3d (16)pCVD442 Ap^(r), sacB (15) pYUB328 Ap^(r), PacI-excisable cosmid vector,ColE1 (5) pYUB405 Ap^(r), Hyg^(r), PacI-excisable cosmid vector, ColE1,does not replicate in (6) mycobacteria pYUB412 Ap^(r), Hyg^(r), E.coli-mycobacteria shuttle PacI-excisable cosmid vector, (6) ColE1origin, int attP, nonreplicative but integration proficient inmycobacteria pYUB601 in vitro repackaged pYUB412::lysA⁺ cosmid frommc²155 library This work pYUB604 4.4-kb EcoRI fragment from pYUB601cloned in the EcoRI site of This work pMV261 pYUB605 5.5-kb NotIself-ligated subclone of pYUB6O4 This work pYUB607 3.4-kb NotI fragmentfrom pYUB604 cloned into NotI site of pKSI⁺ This work pYUB617 7.7-kbinverse XL-PCR product from pYUB604, containing a 1.2-kb This workdeletion of lysA (ΔlysA4) marked with unique SnaBI site. pYUB618 3.2-kbEcoRI fragment from pYUB617, bearing ΔlysA4, blunt cloned This work intoPacI sites of pYUB657 pYUB631 2.5-kb PstI fragment from pCVD442, bearingsacB, cloned into same of This work pMV261 pYUB635 1.3-kb XbaI-BamHIlysA gene from pET3d.lysA, cloned into same sites This work of pKSI⁺pYUB636 3-kb inverse XL-PCR product from pYUB635, containing 95-bp Thiswork deletion of lysA marked with unique MluI site pYUB638 1.4-kb MluIres-aph-res cassette cloned into MluI site in pYUB636 This work pYUB651pYUB412 containing lysA⁺ of M. tuberculosis Erdman, under control of theBCG groEL (Hsp60) promoter pYUB657 3.5-kb NotI-NheI fragment frompYUB631, bearing groEL (Hsp60) This work promoter and sacB, cloned intothe EcoRV site of pYUB405 pYUB659 11-kb SnaBI fragment from cosY373cloned into the EcoRV site of This work pKSI⁺ pYUB665 1.7-kb NheI-Bg/IIfragment from pYUB638 (ΔlysA::res-aph-res) This work replacing 300 bpNheI-Bg/II (lysA⁺) fragment in pYUB659 pYUB667 pYUB665 with the aph generesolved by passage in E. coli DH5α, This work Km^(s) pYUB668 8.4-kbHpaI fragment from pYUB667 cloned into the PacI sites of This workpYUB657 cosY373 pYUB382::M. tuberculosis H37Rv cosmid bearing the lysAoperon (1)

TABLE 3 Electroporation efficiencies and primary recombinationfrequencies for lysA allelic exchange Ave. # Electro- Recombi- SuicideHyg^(r) poration nation Species/strain plasmid (N)^(a) clones^(b)efficiency^(c) frequency^(d) M. smegmatis pYUB618 2 15 ± 3  3 × 10⁵ 5 ×10⁻⁵ mc²155 M. bovis pYUB668 10 5 ± 3 1 × 10⁴ 5 × 10⁻⁴ BCG-Pasteur M.bovis pYUB668 5 2 ± 1 1 × 10³ 2 × 10⁻³ BCG-Connaught M. tuberculosispYUB668 10 3 ± 3 3 × 10⁵ 1 × 10⁻⁵ H37Rv ^(a)(N) = number ofelectroporations for each species/plasmid combination. Each set was donewith the same stock of electrocompetent cells. ^(b)Average number ofHygromycin resistant clones (± standard deviation) from each set ofetectroporations done with the suicide plasmids. ^(c)Electroporationefficiency is the number of Hyg^(r) clones obtained fromelectroporations done with pYUB412, an attP/int Hyg^(r)vector thatintegrates into the attB site of the mycobacterial genome. The number ofHyg^(r) clones from pYUB412 electroporations is an indicator of theelectroporation efficiency of the cells; the number of transformantsobtained with an attP/int vector is equivalent to the # number obtainedwith a replicating vector. We have never observed spontaneous resistanceto hygromycin in the species studied in this paper. ^(d)Recombinationfrequency is calculated by dividing the average number of Hyg^(r) clonesobtained per electroporation with suicide plasmids, divided by theelectroporation efficiency obtained with the vector pYUB412.

TABLE 4 Recombination products from segregation of lysA DUP in differentmycobacterial species Frequency of phenotvpes in Suc^(r) population^(e)(sacB inactivated) (secondary recombinants) Relevant Suc^(r) Hyg^(r)Hyg^(s) Hyg^(s) Species Exp Strain genotype^(a) Media^(b) freq.^(c)(N)_(d) prototrophs prototrophs auxotrophs M smegmatis 1 mc²1492 DUP2 K4 100 67 24 9 2 mc²1492 K 3 100 60 31 9 M. bovis BCG 3 mc²1601 DUP3K,M,T 4 48 2 63 35 Pasteur 4 mc²1602 DUP4 K, M, T 9 46 26 33 41 5mc²1601 DUP3 Basal 0.2 92 9 91 0 6 mc²1601 K 0.9 86 15 73 12 7 mc²1601K, M, T 3 90 11 61 28 8 mc²1601 CAA 6 78 8 92 0 Connaught 9 clone 3 Hom.pYUB688 K N.D. 47 15 51 34 10 clone 9 ″ K N.D. 48 6 54 40 11 clone 10 ″K N.D. 47 10 77 13 12 clone 2 Illeg. pYUB668 K N.D. 48 100 0 0 13 clone4 ″ K N.D. 48 96 4 0 14 clone 8 ″ K N.D. 47 98 2 0 15 clone 11 ″ K N.D.95 100 0 0 M. tuberculosis 17 mc²2998 K 0.3 41 10 90 0 18 mc²2998 K, M,T 1 45 16 84 0 19 mc²2998 CAA 0.6 40 23 77 0 20 mc²2999 Hom. pYUB688Basal 0.5 42 26 74 0 21 mc²2999 K 0.9 38 13 87 0 22 mc²2999 K, M, T 2 4436 64 0 23 mc²2999 CAA (a) 0.7 34 6 94 0 24 mc²2998 Hom. pYUB688 K200 239 44 56 0 25 mc²2998 K200/TW 10 287 20 80 0 26 mc²2998 K1 0.3 96 20 800 27 mc²2998 K1/TW 1 L 96 L 17 L 83 L 0 L 0.8S 63 S 0 S 0 S 100S ^(a)DUPdesignation is used for strains with pYUB688 integrated at lysA withknown orientation (see Table 1). “IlIeg. PYUB688” refers to primaryHyg^(r) Suc^(s) clones in which PYUB688 integrated into the chromosomevia illegitimate recombination. “Hom. pYUB688” refers to primary Hyg^(r)Suc^(s) clones in which pYUB688 integrated at lysA but the orientationof the duplication is unknown. ^(b)Type of media used for outgrowth(Middlebrook 7H9) and sucrose selection (Middlebrook 7H10): Basal (nosupplementation), K (lysine @ 40 μg/ml), K, M, T (lysine, methionine,and threonine each @ 40 μg/ml), CAA (0.2% casamino acids,acid-hydrolyzed), K200 (lysine @ 200 μg/ml), K200/TW (lysine @ 200 μg/mlplus 0.05% Tween-80), K1 (lysine @ 1 mg/ml), K1/TW (lysine @ 1 mg/mlplus 0.05% Tween-80) ^(c)Number of Suc^(r) CFU/ml divided by the viableCFU/ml, (expressed as N × 10⁻⁴). ^(d)(N) = number of Suc^(r) clonesscreened. ^(e)Frequency of phenotypes expressed as a percentage of thenumber of sucrose resistant clones screened. Hyg^(r) prototrophs (notsecondary recombinants-“sacB inactivated”), Hyg^(s) prototrophs(secondary recombinants, wild-type lysA), Hyg^(s) auxotrophs (secondaryrecombinants, ΔlysA). ^(f)For exp. number 27, “L” refers to largecolonies, while “S” refers to small colonies seen on the sucroseselection medium. N.D. (not determined)

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All publications mentioned herein above are hereby incorporated byreference in their entirety. While the foregoing invention has beendescribed in some detail for purposes of clarity and understanding, itwill be appreciated by one skilled in the art from a reading of thedisclosure that various changes in form and detail can be made withoutdeparting from the true scope of the invention in the appended claims.

4 1 28 DNA artificial sequence primer_bind (1)..(28) oligonucleotideprimer used to construct suicide plasmid pYUB618 1 cccgtcgtac gtacgaaccaggttgcgc 28 2 28 DNA artificial sequence primer_bind (1)..(28)oligonucleotide primer used to construct suicide plasmid pYUB618 2cgagtcgata cgtactgctg tgccgccc 28 3 31 DNA artificial sequenceprimer_bind (1)..(31) oligonucleotide primer used to construct suicideplasmid pYUB668 3 gatagcggtc acgcgtctcg tgcgcggtgg a 31 4 32 DNAartificial sequence primer_bind (1)..(32) oligonucleotide primer used toconstruct suicide plasmid pYUB668 4 tccgtacgat acgcgtcagc cacatcggtt cg32

What is claimed is:
 1. A recombinant M. tuberculosis comprising amycobacterial lysA gene containing an unmarked mutation introduced byallelic exchange, wherein the recombinant M. tuberculosis requires amedium containing polyoxyethylenesorbitan monooleate and lysine forgrowth.
 2. The recombinant M. tuberculosis of claim 1, wherein thesource of lysine is casamino acids or casitone.
 3. A method forpreparing a recombinant M. tuberculosis comprising: (a) introducing asuicide plasmid into a M. tuberculosis, said suicide plasmid comprisinga selectable marker, a counterselectable marker, and an unmarked mutantmycobacterial lysA gene; (b) selecting for primary recombinantsincorporating the selectable marker; (c) culturing the primaryrecombinants incorporating the selectable marker; (d) selecting forsecondary recombinants that have lost the counterselectable marker; and(e) isolating the secondary recombinants comprising the desired unmarkedmutant mycobacterial lysA gene to obtain said recombinant M.tuberculosis, wherein said recombinant M. tuberculosis requires a mediumcontaining polyoxyethylenesorbitan monooleate and lysine for growth. 4.The method of claim 3, wherein the selectable marker confers antibioticresistance and the counters electable marker is one of rpsL, pyrF, andsacB.
 5. The method of claim 3, wherein the counterselectable marker issacB.
 6. The method of claim 3, wherein the source of lysine is casaminoacids or casitone.
 7. A recombinant M. bovis BCG comprising amycobacterial lysA gene containing an unmarked mutation introduced byallelic exchange, wherein the recombinant M. bovis BCG grows on a mediumcontaining lysine but does not grow on a medium containing casaminoacids or casitone.
 8. A method for preparing a recombinant M. bovis BCGcomprising: (a) introducing a suicide plasmid into a M. bovis BCG, saidsuicide plasmid comprising a selectable marker, a counterselectablemarker, and an unmarked mutant mycobacterial lysA gene; (b) selectingfor primary recombinants incorporating the selectable marker; (c)culturing the primary recombinants incorporating the selectable marker;(d) selecting for secondary recombinants that have lost thecounterselectable marker; and (e) isolating the secondary recombinantscomprising the desired unmarked mutant mycobacterial lysA gene to obtainsaid recombinant M. bovis BCG, wherein said recombinant M. bovis BCGgrows on a media containing lysine but does not grow on a mediacontaining casamino acids or casitone.
 9. The method of claim 8, whereinthe selectable marker confers antibiotic resistance and thecounterselectable marker is one of rpsL, pyrF, and sacB.
 10. The methodof claim 8, wherein the counterselectable marker is sacB.